Microscope with a viewing direction perpendicular to the illumination direction

ABSTRACT

The invention relates to a microscope, in which a layer of the sample is illuminated by a thin strip of light ( 11 ) and the sample is viewed ( 5 ) perpendicular to the plane of the strip of light. The depth of the strip of light ( 11 ) thus essentially determines the depth of focus of the system. To record the image, the object ( 4 ) is displaced through the strip of light ( 11 ), which remains fixed in relation to the detector ( 8 ), and fluorescent and/or diffused light is captured by a planar detector. Objects ( 4 ) that absorb or diffuse a large amount of light are viewed from several spatial directions. The three-dimensional images, which are captured from each direction can be combined retrospectively to form one image, in which the data is weighted according to its resolution. The resolution of the combined image is then dominated by the lateral resolution of the individual images.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 National Phase Entry Application fromPCT/EP2003/005991, filed Jun. 6, 2003, and designating the UnitedStates.

In contrast to work on single cells, light microscopy studies on embryosand other samples relevant to developmental biology suffer from theparticular problems of absorption and resolution loss. For example,biological questions relating to gene expression patterns in developingorganisms can currently be answered only with difficulty by lightmicroscopy imaging methods, since they are often too slow, too weaklyresolving or technically complex, or they do not allow millimeter-sizeobjects to be viewed from the free working distance or from the sampleholder. An acceptable solution must allow the handling of large samplesand rapid high-resolution acquisition of the data, while beingtechnically as simple as possible to implement.

The scientific literature discloses a microscope for oceanographicresearch, which is characterized in that it produces an illuminationlight plane in a sample chamber by using a laser and detects thefluorescent signals produced in the illumination light planeperpendicularly to this plane by using a camera [E. Fuchs et al., Opt.Express 10, 145 (2002)]. This microscope is similar to theultramicroscope of H. Siedentopf and R. Zsigmondy [Ann. Phys. 10(4), 1(1903)] and is used for the detection of individual free-floatingparticles such as bacteria. It is not suitable for recordingmillimeter-size samples, for example in developmental biology, since acuvette is used as the sample holder. It is likewise unsuitable forthree-dimensional imaging, since it does not have any means of movingthe sample relative to the illumination light plane.

DE 19720513 A1 or U.S. Pat. No. 5,903,781 and the scientific literature[D. Huber et al., J. Microsc. 202, 208 (2001)] disclose an instrumentfor three-dimensional macrography, in which an arrangement for producinglight planes is used for the photographic recording of objects. In thiscase, an object is moved through an illumination plane and the reflectedand scattered light is detected by a camera. This equipment is used toprepare three-dimensional reconstructions of centimeter-size objects. Itis not, however, suitable for the use of fluorescent signals or for thehigh-resolution rendition of objects. A slit pattern diaphragm inconjunction with a mirror arrangement is used for producing the lightplanes. Owing to the use of an only linearly mobile sample stage, thesample cannot be rotated so that it is not possible to view the samplefrom several sides.

The technical scientific literature furthermore discloses constructionsfor optical tomography. Optical projection tomography is used, forexample, in gene expression analysis [J. Sharpe et al., Science 296, 541(2002)]. This is a system in which projections of biological samples arerecorded, the sample being rotated about an axis perpendicular to thedetection direction. Since the sample is not selectively illuminatedperpendicularly to the detection axis by an illumination light plane, incontrast to the microscope according to the invention, the microscopehas a very long depth of focus with which a large part of the sample canbe acquired. The microscope does not therefore offer the opportunity tomove the sample along the detection axis in order to record athree-dimensional image. A three-dimensional image of the sample withspatial resolution is therefore possible only by reconstruction from theprojections.

DE 43 26 473 C2 discloses a confocal theta microscope, which ischaracterized in that it uses a first objective for point illuminationand a second objective to project the object light onto a pointdetector, the detection direction being substantially perpendicular tothe illumination direction. The confocal overlap region of theillumination volume with the detection volume is therefore particularlysmall, and the microscope achieves an almost isotropic resolution whoseorder of magnitude corresponds to the lateral resolution of a confocalmicroscope.

This theta microscope is arranged confocally, however, which placesstringent requirements on the relative alignment of the illumination anddetection focal points. Despite a large working distance, it isfurthermore not readily capable of imaging large objects. This isbecause the object in the theta microscope does not have enough freedomof movement for scanning the object, and owing to the point detection ithas to be scanned in three directions so that imaging takes a very longtime. The illumination light is focused at an illumination point.

It is an object of the present invention to provide a microscope whichis suitable for the high-resolution three-dimensional viewing ofmillimeter-size biological objects, wherein fast acquisition of the datais possible and the structure is technically as simple as possible toimplement.

This object is achieved according to the invention by a microscopehaving at least one illumination beam path and at least one detectionbeam path, characterized in that each illumination beam path is providedwith a focusing arrangement for producing a two-dimensional objectillumination region which extends in the direction of an illuminationaxis of the illumination beam path and transversely thereto, wherein adetection direction of the at least one detection beam path isapproximately orthogonal to the two-dimensional object illuminationregion, and wherein a mobile arrangement is provided for producing arelative movement between the two-dimensional object illumination regionand an object to be studied. The sample is illuminated by a thin lightstrip, and the viewing takes place perpendicularly to this objectillumination region, which has a two-dimensional extent. The thicknessof the illumination light strip thus determines the depth of focus ofthe system to a substantial extent. For imaging, the object is movedthrough the stationary light strip, and fluorescent and/or scatteredlight is recorded by a two-dimensional detector in each position of thescan movement. Since the object can be rotated in the preferredembodiment, it is possible to perform such three-dimensional imagingfrom several sides and combine these to form a single three-dimensionalimage, the resolution of which is essentially determined by the lateralresolution of the individual images. The high resolution of this imageresults from the focused illumination, the perpendicular detection, themovement of the object and the combination of individual exposures byimage processing.

The microscope according to the invention has an illumination light pathand a detection light path, which preferably are mutually orthogonal inthe object illumination region so that the detection direction isperpendicular to the illumination light plane. Nevertheless, theadvantages of the invention are still achieved to a sufficient extentwhen the angle between the illumination and detection directions, orbetween the illumination light plane and the detection direction, doesnot differ too greatly from a right angle.

A laser which allows selective stimulation of fluorescent emission inthe sample is preferably used as the light source. It is preferable touse a cylindrical lens for focusing the illumination light to form athin strip, although it is also possible to use a different focusingelement (for example a holographic element or a conical lens (axicon) ora phase plate or other elements for producing a Bessel beam).

The light which is detected is preferably fluorescent light. It isnevertheless also possible to detect scattered light. The detectionlight is preferably projected onto the detector by a telecentric systemof two objectives. Other optical modules are nevertheless also suitable.

The detection is preferably carried out using a two-dimensional detectorwhich detects the full field, for example a CCD camera. When such adetector is used, rapid imaging is possible and the movement of thesample for a three-dimensional exposure is limited to one direction(i.e. along the detection axis). The resolution of the system isdetermined by the lateral resolution of the detection optics.

Since the surface area of currently available detectors is generallyinsufficient to guarantee complete high-resolution recording of objectsmeasuring several millimeters, in one embodiment of the microscopeaccording to the invention it is possible to move the detector in thedetection plane, i.e. essentially laterally with respect to thedetection direction in order to record images of parts of the object,which can be combined to form an image of the entire object.

In a simple preferred structure, no optical elements are used forguiding the beam paths. Mirrors, dichroic mirrors, beam splitters oroptical fibers, for example, may nevertheless be used for guiding thebeam paths. The fact that the illumination and detection beam paths areseparate in the microscope according to the invention makes it possibleto obviate the use of passive components such as dichroic mirrors oractive, for example acousto-optical components for separating theillumination and fluorescent light, as is customary in otherfluorescence microscopes.

The structure may, for example, be supplemented with a furtherillumination light path, the light of which is focused to form a strip,or object illumination region, which preferably lies in the same planeas the object illumination region of the first illumination light path,so that better lighting of the sample is achieved. The light for thisfurther illumination light path may come from the same light source. Inthis case, the sample is preferably illuminated from two oppositedirections. The alignment workload in the microscope according to theinvention is small in contrast to 4Pi confocal microscopy [S. Hell andE. H. K. Stelzer, J. Opt. Soc. Am., A 9, 2159 (1992)] since it is onlynecessary to superimpose two light strips which are several micrometersthick. Furthermore, the phase of the beams does not need to be takeninto account.

The microscope according to the invention may nevertheless be operatedas a non-confocal 4Pi theta microscope. In this case, as in a 4Pi(A)confocal microscope, the sample is illuminated coherently from twoopposite directions so that an interference pattern which spatiallymodulates the intensity in the illumination light plane occurs alongthis illumination axis. The illumination volume is thereby halved, andby displacing the interference pattern (by adjusting the phasedifference between the beams) it is possible to illuminate complementaryregions of the sample so that an image can be reconstructed withincreased resolution along the illumination axis.

The structure may be supplemented with a further detection light path,for example, by which light emitted in the opposite direction to theexisting detection light path is detected. In this way, detection of thelight can always take place so that the light travels as short aspossible a path through the sample.

Scattered light detectors and/or transmission light detectors mayadditionally be provided.

Although it is possible to place the sample on a sample stage or hold itin air in the microscope according to the invention, the sample ispreferably held from above by a holder in a water-filled sample chamberand can be rotated about the vertical axis, i.e. the axis in thegravitational direction. This has the advantage that there is no changein the gravity acting on the sample, and it is not deformed, when thesample is rotated for imaging from another direction. During such arotation of the sample in the sample chamber, the sample chamber ispreferably not moved so that the optical path lengths (apart fromdifferences due to the refractive index in the sample itself) do notchange during the movement process. This leads to a better imagequality. The sample held in this way may advantageously be oriented soas to minimize the effect of strongly scattering or absorbing parts ofthe sample during the image recording.

In another embodiment of the microscope according to the invention, itis also possible to rotate the illumination and detection paths aboutthe object to be studied while the latter is stationary. Then, however,the sample or object generally needs to be readjusted in order to berecorded in further images.

The object to be studied lies in the two-dimensional object illuminationregion when an image is recorded, the object being substantially largerthan the thickness of this region. A two-dimensional image of the objectparts located in this region is recorded by the two-dimensionaldetector. A three-dimensional image of the object is recorded byscanning the object in the detection direction through the stationaryillumination region (or by scanning the illumination region through theobject), a two-dimensional image being recorded in each position of theobject. The synchronization of movement, illumination and detection ispreferably optimized in order to minimize the stress on the sample.

The rotation of the object (like the linear scan movement) is preferablycontrolled electronically, so that the recording of a plurality ofimages from different angles can be automated and the speed of thesample examination can be increased. The number of images and therotation angles of the sample which are required for recording a fullimage with a particular spatial resolution may be optimized in favor ofa short sample examination time and therefore low stress on the sample.

Preferably, the object to be studied can also be tilted about theillumination axis so that it can also be viewed from additionaldirections. In another embodiment of the microscope according to theinvention, a second detection light path is provided which makes itpossible to detect the light emitted downward. If the objectillumination region is then rotated by 90 degrees about the illuminationaxis (for example by rotating the cylindrical lens), then the sample canbe optically sectioned horizontally (and a three-dimensional image canbe produced by a vertical scan movement).

Advantageously, in the microscope according to the invention, thecylindrical lens can preferably be moved with a high frequency, forexample moved in the illumination light path with a high frequency alongthe cylinder axis and/or the illumination axis, and/or the cylinder axiscan be inclined with a high frequency in the direction of theillumination axis, so that the effect of contamination on thecylindrical lens is less strong and the sample is lit more uniformly.

Preferably, a plurality of biological samples can be held simply bybeing embedded in a gel (about 99% water) or another polymerizing orcrosslinking structure.

The images recorded from different directions by rotating the object tobe studied allow its three-dimensional reconstruction by combining theindividual three-dimensional raw data records. Since only a part of thesample is imaged optimally in the preferred embodiment of the microscopeaccording to the invention (in general the two octants which lie insidethe right angle between the illumination and detection axes), at leastfour images are required for good reconstruction of the full sample.These images can be combined so that the reconstruction offers a higherresolution than the individual images. The quality of the reconstructedimage can be improved by recording along further angles, so that thedead angles of the common optical transfer function are filled in.

When objectives with long focal lengths are used, a working distance ofseveral millimeters is available. The size of the object is primarilylimited by its optical transparency: if all of the object is intended tobe studied (and not just the peripheral layers), enough light from everypart of it must reach the detector in the one or other orientation.

As mentioned above, depending on the magnification of the optical systemin the detection beam path, it may be necessary to displace the detectorassigned to the detection beam path in order to be able to record a fullimage of the object to be studied, essentially owing to the limitednumber of detector pixels in two-dimensional pixel detectors. This meansthat the resolution of the overall system is essentially limited not bythe numerical aperture of the optical components, in particular thelenses being used and therefore the resolution in the sample itself, butrather by the technical limitations which are encountered in the fieldof pixel detectors such as those used, for example, in CCD cameras. Thisproblem may be countered by using high-resolution pixel detectors with apixel number in the range of several millions as those used, forexample, in astronomy or digital photography. These pixel detectors,however, are comparatively expensive and slow.

Another aspect of the present invention therefore relates to amicroscope having at least one illumination beam path and at least onedetection beam path, which is characterized in that each illuminationbeam path is provided with a focusing arrangement for producing a linearobject illumination region which extends in the direction of anillumination axis of the illumination beam path, in that a detectiondirection of the at least one detection beam path is approximatelyorthogonal to the linear object illumination region, and in that atleast one mobile arrangement is provided for producing a relativemovement between the linear object illumination region and an object tobe studied.

According to this aspect of the present invention, the objectillumination region is therefore essentially limited to one dimension,i.e. the longitudinal dimension, so as to change over from thetwo-dimensional structure of the object illumination region as discussedabove to an elongated or linear structure. With this linear objectillumination region, accordingly, only linear sections of an object tobe studied are lit and stimulated to fluoresce, or used to scatterlight. These linear illuminated regions which are now produced can beimaged through the detection beam path or paths onto pixel detectorswhich have an elongated structure, i.e. pixels successively arrangedessentially in one dimension. Such detectors, which are to beinterpreted in principle as “one-dimensional” pixel detectors, can beobtained with a substantially larger pixel number, for example up to8000 pixels. The sections of an object to be studied which are now litby the linear object illumination region and imaged in the detector cantherefore be recorded with a correspondingly high resolution actually inthe detector itself, and correspondingly converted into high-resolutionimages. Since the dimension of the pixels present in such pixeldetectors, transversely to the longitudinal extent of the pixeldetector, is generally much smaller than the width of the image of thelinear object illumination region in this direction, even when takinginto account the magnification produced in the detection beam path, itmay be preferable to change over to elongated “two-dimensional” pixeldetectors which, for example, have a pixel number of 64×4096, i.e. asubstantially larger pixel number in the longitudinal direction of theobject illumination region, or the corresponding image thereof, thantransversely thereto.

In order then to be able to fully image an object to be studied withsuch a system, or to be able to produce a complete image of it, scanningmay be carried out between the object to be studied and the illuminationbeam path or the detection beam path so that, in principle, the objectto be studied is linearly sampled and the individual line images therebyproduced can then be combined to form a full image.

In order to facilitate this, for example, the at least one mobilearrangement may be designed to produce a relative movement between theobject and the linear object illumination region essentiallyorthogonally to the illumination axis and the detection direction. Tothis end, for example, the at least one mobile arrangement may bedesigned to move the object so as to produce a relative movement.

Alternatively or in addition to this displacement of the object to bestudied, the at least one mobile arrangement may be designed to move theat least one illumination beam path at least in the linear objectillumination region provided by it, in order to produce the relativemovement. Since the object illumination region is in this case shiftedwhile the object is stationary, for example, it is then necessary forthe at least one mobile arrangement to be designed to move the at leastone detection beam path in accordance with the movement of the at leastone illumination beam path, at least in its region near the object.

As mentioned above, the at least one detection beam path may have adetector with a multiplicity of detector pixels, in which case it may bepreferable to select the number and positioning of the detector pixelsof the detector so that the at least one detection beam path projects asection of the object, illuminated by the at least one illumination beampath in the object illumination region, essentially fully onto thedetector.

In order to be able to produce not only a surface image of an object tobe studied with the system according to the invention, but also to beable to image it three-dimensionally by three-dimensional sampling, itis furthermore proposed for the at least one mobile arrangement to bedesigned to move the object to be studied, essentially in the directionof the detection direction of the at least one detection beam path.

From the explanations above, it is clear that an essential principle ofthe present invention is to produce an elongated object illuminationregion with at least one illumination beam path, in which an object tobe studied can then be positioned in order to produce an image of thesection of the object to be studied positioned in the objectillumination region, or optionally the entire object, by stimulatingfluorescence or by generating scattered light in at least one detectionbeam path.

The present invention therefore also relates to a microscope which hasat least one illumination beam path and at least one detection beampath, and is characterized in that each illumination beam path isprovided with a focusing arrangement for producing an objectillumination region which extends in the direction of an illuminationaxis of the illumination beam path, in that a detection device of the atleast one detection beam path is approximately orthogonal to the objectillumination region, and in that a mobile arrangement is provided forproducing a relative movement between the object illumination region andan object to be studied.

The invention will be explained in more detail below with reference tothe appended drawings, in which:

FIG. 1 shows the schematic representation of the beam path in oneembodiment of the microscope according to the invention, a singleillumination beam path and a single detection beam path being provided,seen in observation direction I of FIG. 2;

FIG. 2 shows the embodiment represented in FIG. 1 in observationdirection II in FIG. 1;

FIG. 3 shows an outline representation of the illumination beam path,which emerges from a cylindrical lens and forms an object illuminationregion in the region of a focus line;

FIG. 4 shows a plan view of the beam path of FIG. 3 in observationdirection IV in FIG. 3;

FIG. 5 shows the schematic representation of the beam path in anotherembodiment of the microscope according to the invention, twoillumination beam paths being provided;

FIG. 6 shows another outline representation of a microscope according tothe invention;

FIG. 7 shows a view corresponding to FIG. 1 of an alternativelyconfigured microscope according to the invention.

FIG. 1 shows an embodiment of a microscope 100 according to theinvention. The structure comprises a light source 1, the collimatedlight beam 2 from which is focused into the sample 4 by a cylindricallens 3. This creates a thin vertical light strip 11 by which fluorescentemission can be induced in the sample 4. The emitted light 5 isprojected through detection optics 6 onto the two-dimensional detector8, for example a CCD camera.

The structure is particularly simple owing to the right-angledarrangement (=90 degrees) of the illumination direction 9 and thedetection direction 10. In particular, the use of dichroic mirrors forseparating the illumination and fluorescent light in the detection beampath 5 can be obviated. The filters 7 in the illumination beam path 2and in the detection beam path 5 are glass filters oracousto-/electro-/magneto-optical filters, and allow selective choice ofwavelengths for the illumination and the detection.

The sample 4 is held in a sample chamber 13 by a holder 12, and is movedthrough the stationary light plane 11 in the detection direction 10 forimaging. The holder 12 also makes it possible to rotate the sample 4about its vertical axis 14, so that the sample 4 can be illuminated andviewed from several sides.

FIGS. 3 and 4 show in outline the aforementioned illumination beam path2 which is produced with the aid of the cylindrical lens 3. Using thecylindrical lens 3, whose focal length may preferably lie in the rangeof from 10 mm to 100 mm, light emitted by the light source 1 is focusedat a comparatively small angle α. In the region of a focus line L, thiscreates an object illumination region 20 indicated by dashes in FIG. 3,which approximately has a two-dimensional or flat structure or extent,and is formed by cylinder sections on either side of the focus line.With a dimension a of about 5 mm for this object illumination region 20as measured in the direction of the illumination axis, or illuminationdirection, and with a thickness dimension of about 20 μm for theillumination beam path 2 in the region of the focus line b, a thicknessdimension c of approximately 60 μm is obtained at the end regions 22, 24of the object illumination region 20 lying in the illuminationdirection, although this naturally depends on the numerical apertureprovided for the cylindrical lens 3. Throughout the object illuminationregion 20, there is therefore a negligible variation—expressed in termsof the dimensions of the objects to be studied—in the thickness of theobject illumination region 20 in the illumination beam path 2, so that,in particular taking into account the dimensions of the objects to bestudied, a constant thickness of the object illumination region andtherefore a two-dimensional or planar structure thereof can be assumedhere to a first approximation.

FIG. 5 represents a modified configuration of the microscope 100, inwhich two illumination beam paths 2, 2′ are provided. In the caserepresented, each of these two illumination beam paths 2, 2′, which haveoppositely directed illumination directions but illumination axescorresponding to each other, respectively has a cylindrical lens 3, 3′with optionally assigned filters 7, 7′ and a light source 1, 1′. In avariant of this configuration, it is furthermore possible to providemore than just one light source. By superposition of the two objectillumination regions of these illumination beam paths 2, 2′, whichobject illumination regions were presented in more detail above withreference to FIGS. 3 and 4, this creates a thin vertical light stripwhich is more homogeneous compared to the light strip in the embodimentrepresented in FIG. 1. The emitted light 5 is projected throughdetection optics 6 onto the two-dimensional detector 8. This embodimentof the microscope according to the invention is particularly suitablefor absorbent samples, for which it is not possible to illuminate theentire sample with illumination from one side.

In this configuration, it is possible to make the two illumination beampaths 2, 2′, or their light beams, interfere deliberately by definedadjustment of the phase angle of these light beams with respect to eachother, wherever the two object illumination regions of these twoillumination beam paths 2, 2′ mutually overlap. In this way, in theregion where the object or sample 4 to be studied is to be illuminated,it is possible to stop out particular sections by destructiveinterference or highlight particular regions by constructiveinterference, so that the resolution of the overall system can befurther improved.

FIG. 6 depicts another variant of the microscope 100 according to theinvention. The arrow P indicates that the cylindrical lens 3 showntherein can be rotated, for example through 90°, about the illuminationaxis of the illumination beam path 2. The object illumination region 20of this illumination beam path 2 is thereby also rotated so that,starting from the orientation shown in FIG. 2 in which it liesessentially in the plane of the drawing, it is rotated through 90° andis then perpendicular to the plane of the drawing. In this way, it ispossible to view the object 4 to be studied from a different direction,i.e. the direction lying below this object 4 in the representation ofFIG. 2. It is also possible to provide a further detection beam path 5′with which, in relation to the detection beam path 5 visible in FIG. 1,the object 4 to be studied can be viewed at an angle of 90° without thisobject 4 itself having been rotated.

In such a system, for example by using mirrors 60 and a tilting mirror26, it is possible to guide different detection beam paths 5, 5′selectively to the same detector 8, or the same optical system withobjectives 6, according to the position of the tilting mirror 26. Inaccordance with the rotational position of the cylindrical lens 3, thetilting mirror 26 will then correspondingly be switched over. It is ofcourse possible to provide two detection beam paths 5, 5′ with arespectively assigned objective arrangement and detector, independentlyof each other and for example at an angle of 90°. It is also possible tomake at least one of these systems mobile such that it can be rotatedtogether with the cylindrical lens 3 about the illumination axis of theillumination beam path 2 in FIG. 2, so that imaging can then be producedall around the object 4 to be studied by simultaneously rotating thecylindrical lens 3 and this detection beam path, without this objectitself having been moved.

An alternatively configured microscope 100 according to the invention isrepresented in FIG. 7. The basic structure, as well as the viewing mode,are the same as in FIG. 1 so that reference may generally be made to thecomments above. There is again an illumination beam path, essentiallyprovided by the light beam 2, which is focused by using an opticalsystem in order to generate an object illumination region 20′. The lens3 used here is no longer a cylindrical lens, but a lens which isrotationally symmetric with respect to the illumination axis 9 of theillumination beam path. This generates an object illumination region 20′which is likewise essentially rotationally symmetric and therefore,particularly in the region where the object 4 to be studied i.e. thesample is positioned, can be interpreted as a linear object illuminationregion or line-like object illumination region. The dimensions of thisessentially rotationally symmetric linear object illumination regionmay, naturally depending on the focal length of the lens 3 or of theoptical system being used, be in the range of 10-20 μm at the centerwhile having a dimension in the range of 40-60 μm in the edge regions.

In this microscope 100 as shown in FIG. 7, therefore, a correspondinglylinear region instead of a wafer-like or two-dimensional region of thesample 4 is lit when a sample 4 is positioned in the object illuminationregion 20′. The image of this linearly illuminated region as produced inthe detection beam path 5 is projected via its optical system onto thedetector 8, so that an optionally magnified line image is generatedthere. The detector 8 is constructed as a pixel detector and, inaccordance with the linear image now produced, has more of a“one-dimensional” pixel arrangement. In this arrangement, thepositioning of the pixels is such that a substantially larger number ofthe pixels will lie successively in the longitudinal direction of thelinear image which is produced, than transversely thereto. The number ofpixels in the longitudinal direction or transverse direction ispreferably selected so that the linear image produced in the detectionbeam path 5 can be recorded fully by the detector 8 without having tomove the latter. In the extreme case, it is even possible to provide asingle row of pixels. Here, it should be borne in mind that with adecreasing width of such a pixel array and progressive transition to asingle pixel line, the number of pixels present in such a lineararrangement can be increased and hence the resolution of the detector 8can be enhanced correspondingly in this direction.

In order to be able to produce full images of the sample 4 with thishigh-resolution microscope 100 as represented in FIG. 7, even in view ofthe possible structure of the detector 8, a relative movement of thissample 4 with respect to the linear object illumination region 20′ isproduced according to the invention. There are in principle various waysof doing this. On the one hand, the sample 4 may be displacedorthogonally to the linear object illumination region and alsoorthogonally to the detection beam path 5, i.e. perpendicularly to theplane of the drawing in the representation of FIG. 7. In this way,linear regions of the sample 4 are successively illuminated by scanning,so that a full image of the sample 4 in a plane can be produced bycombining the correspondingly recorded images. In order to generate athree-dimensional image, the sample may furthermore be displaced in thedirection of the detection beam path 5 or the detection direction 10, sothat various planes of the sample 4 are moved through the focal plane ofthe illumination beam path 5.

In an alternative variant represented in FIG. 7, the sample 4 may bekept stationary while providing devices 24 and 26 both in theillumination beam path 2 and in the detection beam path 5 whichfacilitate displacement of these two beam paths 2, 5 in their regionnear the sample. For example, these devices 24, 26 may be beamdeflection units which, for example, respectively contain a tiltablemirror. The two beam deflection units 24, 26 are operated by a controldevice 28 in order to match their movements to each other, which ensuresthat the region of the sample 4 currently being lit by the objectillumination region 20′ is always projected through the detection beampath 5 onto the detector 8. In this way it is possible for the sample,which otherwise cannot be moved perpendicularly to the plane of thedrawing in FIG. 7, to be scanned in a plane. Here again, in order toobtain a three-dimensional representation, the sample 4 is thendisplaced again in the detection direction 10 in order to sample aplurality of planes in succession.

It is of course also possible to combine the two aforementioned ways ofproducing a relative movement between the sample 4, on the one hand, andthe beam paths, on the other hand, in which case it should be borne inmind that the movement of the beam paths can be carried out faster thanthe movement of the sample.

With the system as represented in FIG. 7, it is thus possible to usehigh-resolution “one-dimensional” or elongated pixel arrays in thedetector 8 in order to be able to produce correspondinglyhigh-resolution images of the respectively lit regions of the sample 4.A further advantage of this “sampling” of the sample 4 is that theposition of the object illumination region can be matched better to thefield of view of the detection optics, i.e. regions which are notintended to be imaged do not have to be sampled or illuminated at all,without causing any deterioration of the sampling or imaging of otherregions of interest. This scan movement of the linear objectillumination region with respect to the sample to be studied furthermoregradually generates an illumination plane or a two-dimensional objectillumination region which has a more homogeneous intensity in thedirection of the relative movement than is the case with correspondingbeam expansion in the systems described above, since the intensityprofile in the object illumination region therein is also dependent onthe intensity profile of the focused light beam. Higher intensities canalso be achieved owing to the stronger focusing of the light. The laserpower is therefore used more efficiently, which is advantageousparticularly for weak fluorescence or multi-photon stimulation.

It should be pointed out that various aspects of the microscopeaccording to the invention, for example the provision of differentnumbers of illumination beam paths and detection beam paths, or therelative positioning and phase adjustment thereof, the measures formoving an object by displacement or rotation or for moving the opticalsystem, for example while the object is stationary, may of course beimplemented irrespective of whether the object illumination region has atwo-dimensional extent or is formed essentially as a band or linearly.

The invention relates to a microscope in which a layer of the sample isilluminated by a thin light strip 11 and the viewing takes placeperpendicularly to the plane of the light strip. The thickness of thelight strip 11 therefore essentially determines the depth of focus ofthe system. For the imaging, the object 4 is moved through the lightstrip 11 which is stationary with respect to the detector, andfluorescent and/or scattered light is recorded by a two-dimensionaldetector. Strongly absorbing or strongly scattering objects 4 are viewedfrom several spatial directions. The three-dimensional images which arerecorded from each direction can subsequently be combined to form animage in which the data are weighted according to their resolution. Theresolution of the combined image will then be dominated by the lateralresolution of the individual images.

1. A microscope having at least one illumination beam path and at leastone detection beam path, characterized in that each illumination beampath is provided with a focusing arrangement for producing a linearobject illumination region which extends in a direction of anillumination axis of the illumination beam path, a detection directionof the at least one detection beam path is approximately orthogonal tothe linear object illumination region, and at least one mobilearrangement is provided for producing a relative movement between thelinear object illumination region and an object to be studied, whereinthe linear object illumination region is essentially limited to thedirection of the illumination axis.
 2. The microscope as claimed inclaim 1, characterized in that the at least one mobile arrangement isdesigned to produce a relative movement between the object and thelinear object illumination region essentially orthogonally to theillumination axis and the detection direction.
 3. The microscope asclaimed in claim 2, characterized in that the at least one mobilearrangement is designed to move the object in order to produce arelative movement.
 4. The microscope as claimed in claim 2,characterized in that the at least one mobile arrangement is designed tomove the at least one illumination beam path at least in the linearobject illumination region provided by it, in order to produce therelative movement.
 5. The microscope as claimed in claim 4,characterized in that the at least one mobile arrangement is designed tomove the at least one detection beam path in accordance with themovement of the at least one illumination beam path, at least in itsregion near the object.
 6. The microscope as claimed in claim 1,characterized in that the at least one detection beam path has adetector with a multiplicity of detector pixels.
 7. The microscope asclaimed in claim 6, characterized in that the number and positioning ofthe detector pixels of the detector are selected so that the at leastone detection beam path projects a section of the object, illuminated bythe at least one illumination beam path in the object illuminationregion, essentially fully onto the detector.
 8. The microscope asclaimed in claim 1, characterized in that the at least one mobilearrangement is designed to move the object to be studied essentially inthe direction of the detection direction of the at least one detectionbeam path.